Background: The Repeat Expansion Diseases are caused by intergenerational expansionsof a specific tandem repeat. More than 20 such diseases that belong to this group havebeen identified thus far. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. Carriers of alleles with 55-200repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder,Fragile X-associated tremor/ataxia syndrome (FXTAS) and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency (FXPOI). Furthermore, in females,the PM allele can undergo expansion on intergenerational transfer that can result in their children having alleles with much larger numbers of repeats. Such full mutation (FM) alleles become epigenetically silenced via a process we are still trying to understand. Silencing results in a deficiency of the protein product of this gene, FMRP,which is involved in, amongst other things, insulin signaling and glucose metabolism. The FMRP deficiency results in Fragile X syndrome, the most common heritable cause of intellectual disability and autism. The threshold for methylation is currently considered to be 200 repeats based on Southern blotting of DNA from individuals symptomatic for FXS. Progress report: In this reporting period we used a FX patient-derived embryonic stem cell (ESC) line to study the mechanism of gene silencing that is responsible for FXS. We showed that silencing was already present in these cells. This suggests that silencing can occur in the very early embryo and need not depend on late events in differentiation as has been previously suggested. In addition, we observed that while expansions were not seen in these cells, contractions were. This, together with our previous observations that expansions only occur when the FMR1 gene is transcriptionally active, adds weight to the idea that expansions and contractions occur by different mechanisms. Furthermore, we found that when these contractions resulted in alleles that had >400 repeats, silencing was retained. However, when the repeat number dropped below 400, then silencing was rapidly lost. In the now transcriptionally active ESCs, resilencing was not observed even after differentiation in neurons. These results have important implications. Firstly, it is consistent with the emerging idea in the field that silencing in the early embryo is dynamic when cells are rapidly dividing, the expression of demethylating enzymes is high and the level of the maintenance methylase in the nucleus is low. Secondly, it suggests that, at least in these cells, that the threshold for methylation is 400 repeats. A retrospective examination of the literature showed that many carriers of unmethylated FM alleles had alleles with <400 repeats. Thus, rather than having second site mutations that prevent methylation as has been previously suggested, the 200 repeat threshold for methylation typically used in the field may need to be revised upwards. In addition to our work on gene silencing, we have also extended our previous work on the pathology seen in a mouse model of the FXDs to show that these animals have mitochondrial defects that could contribute to both FXTAS and FXPOI. This is significant since these mice do not make the toxic Repeat-associated Non-AUG (RAN) protein that is made in human PM carriers and in other mouse models. Thus, our data support the idea that the FMR1 transcript may indeed have intrinsically deleterious effects independent of the ability to make this abnormal protein. We have also identified factors that affect the hyper-expression of the PM allele. Future work will focus on developing a deeper understanding of the relevance of these factors for the etiology of FXTAS and FXPOI.